Degradation Of Extracellular Antibiotic Resistance Genes With Uv254 Treatment

Disinfected wastewater effluent contains a complex[GRAPHIC]mixture of biomolecules including DNA. If intact genes conveying antibiotic resistance survive the disinfection process, environmental bacteria may take them up. We treated plasmid pWH1266, which contains ampicillin resistance gene (TEM)-T-bla-1 and tetracycline resistance gene tetA, with UV254 doses up to 430 mJ/cm(2) and studied the ability of those genes to be acquired by Acinetobacter baylyi. The plasmids required approximately 20-25 mJ/cm(2) per log(10) loss of transformation efficiency. We monitored plasmid DNA degradation using gel electrophoresis and qPCR with both short amplicons (similar to 200 bps, representative of ARG amplicon lengths commonly used for environmental monitoring) and long amplicons (800-1200 bps, designed to cover the entire resistance genes). The rate of transformability loss due to UV254 treatment was approximately 20x and 2x larger than the rate of gene degradation measured with the short and long amplicons qPCR, respectively. When extrapolated to account for the length of the entire pWH1266 plasmid, the qPCR rate constants were 2-7x larger than the rate constants measured with transformation assays. Gel electrophoresis results confirmed that DNA cleavage was not a major inactivating mechanism. Overall, our results demonstrate that qPCR conservatively measures the potential for a gene to be transformed by environmental bacteria following UV254 treatment.